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Improved negative-strand RNA viral vector

Pending Publication Date: 2018-07-12
ID PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention enhances the removal of a vector in iPS cells by adding a degron to the P protein. This results in high gene expression and rapid removal of the vector without the need for high temperature cultivation. Additionally, it regulates transcription factor expression and enhances vector removal in production of iPS cells.

Problems solved by technology

However, P protein-deficient vectors have problems of low expression of carried genes and short-term expression (high expression capability of SeV vectors is impaired), and conventional temperature-sensitive mutant vectors have a problem in that it takes time to remove the vectors (culturing for several days at 39° C.).
Incidentally, mir-302 is not a versatile method since expression thereof varies depending on cell species, and siRNA is a method that is influenced by gene transfer efficiency and inhibitory efficiency.

Method used

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Examples

Experimental program
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Effect test

example 2

[0170]HeLa cells were seeded into 12-well plates at 1×105 cells / well. SeV(F)ddAG / TSΔF (ddAG) was infected at MOI=10 at 37° C. (day 0), 1 μM of Shield1 was added thereto on day 1, and after 5 hr. observation with a fluorescence microscope and analysis by FACS were carried out. FACScalibur (BD Biosciences) was used as FACS. As a result, green fluorescence was observed in ddAG infected cells added with Shield and strong fluorescence of ddAG+Shield1 was observed with FACS. It was difficult to determine fluorescence of ddAG not added with Shield1 under the microscope, but in FACS, fluorescence at the base level that is clearly discerned from control cells which were not infected with ddAG was observed (FIG. 2).

example 3

[0171]HeLa cells were infected with SeV(HNL)ddAG / TSΔF (ddAG) at MOI=10 at 37° C. (day 0), 1 μM of Shield1 was added thereto on day 1, and after 5 hr, observation with a fluorescence microscope and analysis by FACS were carried out. As a result, green fluorescence was observed in ddAG infected cells added with Shield1 and strong fluorescence of ddAG+Shield1 was observed with FACS. Fluorescence of ddAG not added with Shield was decreased by carrying the gene at HNL position as compared to SeV(F)ddAG / TSΔF on which the gene was carried at F position, but with FACS, fluorescence at the base level that is clearly discerned from control cells which were not infected with ddAG was observed (FIG. 3).

example 41

[0172]HeLa cells were infected with SeV(HNL)d1GFP / TSΔF (d1GFP), SeV(HNL)d2GFP / TSΔF (d2GFP), SeV(HNL)d4GFP / TSΔF (d4GFP), SeV(HNL)d1AG / TSΔF (d1AG), SeV(HNL)d2AG / TSΔF (d2AG), and SeV(HNL)d4AG / TSΔF (d4AG) at MOI=10 at 37° C. (day 0), and then observation with a fluorescence microscope was carried out on day 2. As a result, a decrease in fluorescence was observed by adding a PEST sequence to the C terminus side of GFP or AG Among d4GFP, d2GFP, and d1GFP, fluorescence of d4GFP was strong and fluorescence of d1GFP was weak (FIG. 4). It was possible to observe d2AG and d4AG under the fluorescence microscope, but it was difficult to determine d1AG under the fluorescence microscope. Thus, in the subsequent tests, d2 and d4 were used.

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Abstract

The present invention addresses the problem of providing an improved negative-strand RNA viral vector enabling transient high expression of a gene carried by the vector, and quick removal of the vector after the expression, and the use thereof. It was found that if a degron is added to a P-protein possessed by a negative-strand RNA viral vector, high-level expression of a gene carried by the vector is transiently induced after introduction of the vector, and thereafter, the vector can be quickly removed in a manner dependent on the degron. In particular, if the degron is added to a temperature-sensitive P-protein, the vector can be removed to a level below the detection limit within two weeks after cells are infected with the vector. Since the present invention is useful for transiently expressing a transcription factor, such as a reprogramming factor or the like, in target cells, and then quickly removing the vector, the present invention is expected to be applied in cell therapy and regenerative medicine.

Description

TECHNICAL FIELD[0001]The present invention relates to an improved negative-strand RNA virus vector. More specifically, the present invention relates to a negative-strand RNA virus vector including the P protein added with a degron and a use thereof.BACKGROUND ART[0002]Negative-strand RNA virus vectors such as Sendai virus (SeV) vectors are cytoplasm-type RNA virus vectors (vectors whose all expression stages are carried out in the cytoplasm), and thus, even when such negative-strand RNA virus vectors are used in vivo, there is no concern in that carried genes are integrated into host chromosomes to generate genetic toxicity. Furthermore, the negative-strand RNA virus vectors have various excellent capabilities, for example, high gene transfer efficiency and high expression efficiency can be obtained in both in vitro and in vivo and long-term persistent expression in vitro can be achieved. For these reasons, SeV vectors are widely applied and used as gene transfer vectors in producti...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/04
CPCC12N15/85C12N7/04C12N2760/18843C12N2830/42C12N2760/18861C12N2830/60C12N15/86C12N2760/18821C12N2760/18822
Inventor SAEKI, KOICHI
Owner ID PHARMA
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